Role of the BAF180 remodelling complex in transcriptional repression and DNA double strand break repair in mammalian cells.

DNA is the genetic material which encodes the information that determines the ability of our cells to function. DNA is assembled from building blocks called base pairs, and the sequence of the base pairs is used to create the enzymes and proteins that carry out the work within a cell. Since each individual originates from a single cell, the genetic material of each cell in a body should be identical. However, cells have to go through multiple divisions to generate an organism, which necessitates that our DNA is replicated accurately. Additionally, our DNA is constantly subjected to damage, from both external and internally generated damaging agents. To maintain genetic stability, that is, the correct sequence of base pairs in our DNA, which is critical for the health of the individual and for cancer avoidance, cells have a repertoire of mechanisms that serve to repair any damage to the DNA during replication or from DNA damaging agents. A particularly dangerous lesion is when a break occurs in both DNA strands in close proximity. Such a lesion is called a DNA double strand break (DSB). If a DSB remains unrepaired, the cell will be unable to replicate and can die but, as potentially damaging, if the DSB is misrepaired, the sequence of base pairs can become changed, which is a step in the aetiology of carcinogenesis. DNA is embedded in a coat of proteins called histones, which collectively generate a protective shield to the DNA called chromatin. However, chromatin can be refractory to the repair processes. Additionally, a range of metabolic processes take place on the DNA, which can also inhibit the repair process. The genetic information encoded in the DNA is employed to generate proteins, the worker molecule of a cell. Transcription is the first step by which proteins are assembled from the genetic sequence, and transcription can be inhibitory to the DSB repair process. Thus, the cell has evolved processes that allow the chromatin to be remodelled to facilitate DSB repair and that allow transcription in the vicinity of a DSB to be inhibited. Although, we have a good understanding of the core DSB repair processes, we have very little understanding of how chromatin becomes remodelled to facilitate repair to occur nor how the repair process interface with processes such as transcription. In preliminary work, we have made the exciting observation that the PBAF complex is required for the inhibition of transcription in the vicinity of a DSB and that PBAF is required for a subcomponent of DSB repair. The aim of this proposal is to learn more about how PBAF impacts upon transcription at a DSB and how it influences the DSB repair process. This is important because a component of the PBAF complex, called BAF180, is frequently mutated in cancers of the kidney. This suggests that PBAF protects the integrity of our DNA. Thus, understanding how it impacts upon the process of DSB repair and transcription could help us understand how we are protected against cancer.

Chromatin profoundly impacts on DNA damage responses and other DNA transactions with a cascade of chromatin alterations arising during DNA repair and processes such as transcription. Active genes in the vicinity of DNA double strand breaks (DSBs) are repressed in an ATM-dependent manner, but changes to chromatin structure, including the role of chromatin remodelling enzymes, remain largely uncharacterised. Failure to accurately repair DSBs, particularly in transcriptionally active euchromatin, can lead to chromosomal aberrations and genome instability. We will investigate the mechanism by which chromatin is reorganized to facilitate accurate repair of DNA DSBs in transcriptionally active regions of chromatin.
We have discovered that the PBAF chromatin remodelling complex is required for DSB-dependent transcriptional repression. We will investigate the mechanism by which PBAF mediates this function. Using human cell lines, we will monitor DSB repair by assessing g-H2AX foci formation. H2AX is a variant form of the histone H2A and becomes phosphorylated in the vicinity of DSBs. We will develop a ChIP assay to monitor events at a DSB in a transcriptionally active region. This will be exploited to determine the timing and genetic dependence of PBAF recruitment to transcriptionally active DSBs, and to analyse changes to chromatin structure and composition. The BAF180 subunit of PBAF, which is frequently mutated in cancer, is critical for PBAF's role at DSBs. We will undertake a functional analysis of BAF180 using site directed mutagenesis to examine regions of BAF180 required and the molecular mechanisms involved. Also, we will determine whether PBAF-mediated transcriptional repression prevents chromosomal instability by analysing translocation formation. These studies will reveal the mechanisms that control the cross-talk between DSB repair and transcription. Ultimately, this information will lead to greater insight into the biology of tumourigenesis.

This project will investigate the role of PBAF in mediating DNA damage responses and promoting genome stability. Genes encoding subunits of PBAF (one of the two SWI/SNF complexes in mammalian cells) are frequently mutated in cancer. Strikingly, the gene encoding BAF180, which we determined is critical to PBAF's role in mediating DSB-dependent transcriptional repression, is mutated in over 40% of renal cell carcinomas (Varela et al Nature 2011). Generating greater insight into the function of PBAF in mediating DNA repair has the potential to impact in the following ways:
1) To identify new diagnostic or therapeutic targets for use in the clinic with cancer patients.
2) To provide a greater understanding of human disorders caused by defects in DNA DSB repair, including LIG4 Syndrome, XLF deficiency and Artemis deficiency.
3) To provide enhanced understanding of human disorders that impact upon chromatin architecture, most importantly CHARGE Syndrome, which has mutations in CHD7, a component of the PBAF complex.
4) An enhanced understanding of how DSBs impact upon transcription could enhance our understanding of the impact of radiation exposure and be of interest to regulators.